A common feature in a number of autoimmune diseases and inflammatory conditions is the involvement of pro-inflammatory CD4+ T cells. These T cells are responsible for the release of inflammatory Th1 type cytokines. Cytokines secreted by CD4+ Th1 cells include IL-2, IFN-γ, TNF-α and IL-12. These pro-inflammatory cytokines stimulate the immune response and can result in the destruction of autologous tissue. Th2 cytokines are associated with suppression of T cell response, and include IL-10, IL-4 and TGF-β. Th2 cytokines have been used to suppress immune responses and to treat or prevent autoimmune diseases, such as type 1 diabetes and multiple sclerosis.
Type 1 diabetes (T1D, a.k.a. insulin dependent diabetes mellitus (IDDM)) is caused by T cell-mediated autoimmune destruction of insulin-producing β cells in the pancreatic islets. Analysis of the immune response to β-cell antigens has shown that both CD4 and CD8 T-cells contribute to β-cell deletion, through mechanisms dependent on pro-inflammatory cytokines such as IL-12 and IFN-γ that support a typical ‘Th1’ response. The non-obese diabetic (NOD) mouse is a well-recognized model for human IDDM. Female NOD mice develop spontaneous diabetes from approximately 20-30 weeks of age, following a pre-diabetic phase consisting of non-pathogenic autoantibody production, and peri-islet mononuclear cell infiltration, which develops at around 10-12 weeks of age. At 30 weeks of age, more than 80% of female mice have developed overt diabetes (hyperglycemia).
The ability to identify individuals at risk for IDDM, through detection of anti-β-cell autoantibodies prior to the onset of overt clinical disease, offers the potential to introduce immunotherapies that may subvert the development of a cell mediated response, resulting in a lack of progression to overt clinical symptoms. The classification of IDDM as a ‘Th1’ cell mediated response has lead to the development of immunotherapies that support the induction of counter regulatory networks dependent on the induction of ‘Th2’ immunity to β-cell antigens. As mentioned above, Th1 responses are dependent on the secretion of IL-12 from antigen presenting cells, and IFN-γ from effector T-cells. IL-4 is a natural regulator of the Th1 compartment, and several studies employing systemic delivery of IL-4 have shown that this approach can prevent the onset of clinically overt diabetes in the NOD mouse model.
Multiple intraperitoneal (i.p.) injections of IL-4, when initiated at an early age, have been shown to prevent IDDM in NOD mouse models by producing a suppressive response in Th2 type T cells. For example, Rapaport et al. (J Exp Med (1993) 178:87-89) disclose that i.p. administration of recombinant IL-4 (twice weekly for 14 weeks) to prediabetic NOD mice beginning at 6 weeks of age reduced the incidence of IDDM to less than 10% compared to greater than 75% in control animals. Cameron et al. (J Immunol (1997) 159:4686-4692) confirmed this study and disclose that i.p. administration of recombinant IL-4 beginning at an earlier age (3 times a week for 10 weeks beginning at 2 weeks of age) improved protection.
As an alternative to injection of recombinant IL-4, gene therapy vectors have been employed, using both naked DNA and viral vector approaches (Chang and Prud'homme (J Gene Med (1999) 1:415-423) and Cameron et al. (2000 Human Gene Therapy 11:1647-1656)). Chang et al. administered a naked DNA plasmid encoding an IL-4/IgG1 chimeric protein with IL-4 activity by intramuscular injection to 3 or 6 week old NOD mice every 21 days for five administrations. Prolonged expression of the IL-4 fusion protein was detectable in muscle at days 7 and 21. The best protection against diabetes was obtained by injecting five doses of DNA at three-week intervals. Chang and Prud'homme concluded that delivery of constant, but low, cytokine levels over a relatively long period would be advantageous.
Cameron et al. compared the efficacy of two IL-4 DNA vectors, with and without an EBV origin for episomal replication, for the prevention of diabetes in NOD mice. Three biolistic epidermal inoculations of NOD mice (at 3, 5 and 7 weeks of age) with either DNA vector resulted in a reduction of insulitis and diabetes. Production of IL-4 (40-50 pg/ml) by the vector lacking an episomal origin of replication was observed in sera of treated NOD mice, but was not detectable at time points later than 3 days post inoculation. In contrast, the serum levels of mIL-4 produced by the vector containing the EBV origin of replication were higher (50-100 pg/ml) and were detectable at 12 days post-inoculation. At 30 weeks of age, 45% of mice treated with the non-replicating IL-4 vector, and 20% of the mice treated with the episomally maintained vector were diabetic, as compared to 90% of control mice. Cameron et al. observed that due to the short half life of IL-4 in vivo, multiple injections of this cytokine are required to protect NOD mice from IDDM (i.e., thrice weekly for 8-10 weeks). They concluded, “Cytokine immunotherapy with the intent to induce immune deviation is most effective in preventing the pathogenesis of T1D when initiated at an early age and maintained at low doses continuously during the prophylactic period.”
Cameron et al. (Gene Therapy (2000) 7:1840-1846) treated NOD mice with two i.p. injections of recombinant replication deficient adenovirus type 5 vector expressing murine IL-4 (Ad5mIL-4) beginning at two weeks of age. This treatment delayed and reduced the incidence of diabetes from 80% in controls to 20% in Ad5mIL-4 treated mice. In Ad5mIL-4 treated mice, the onset of diabetes was delayed until 28 weeks of age, while in control mice, diabetes was observed as early as 14 weeks of age. IL-4 (1-2 ng/ml) was detectable in the serum of treated NOD mice for up to 3 days following each injection at 2 and 5 weeks of age, yet was undetectable after a third injection at 7 weeks. However, a single injection at 2 weeks of age did not reduce the incidence of IDDM in NOD mice, but resulted in a 10-week delay in onset as compared to controls. Interestingly, delaying administration of the gene therapy vector until mice were 5 weeks of age did not reduce diabetes incidence in later life.
Lee et al. (Pharmaceutical Res (2002) 19:246-249) administered an IL-4 expression plasmid complexed with a biodegradable carrier by a single i.v. injection to 4-week-old NOD mice. Exogenous IL-4 expression was detectable in the liver five days post injection and the severity of insulitis was reduced at 10 weeks of age.
Mueller et al. (JEM (1996) 184:1093) demonstrated that transgenic NOD mice expressing IL-4 in their pancreatic β cells under the control of the constitutive human insulin promoter are completely protected from insulitis and diabetes.
Feili-Hariri et al. (Diabetes (1999) 48:2300-2308) disclose that bone marrow derived dendritic cells (DCs) prepared in GM-CSF and IL-4, and adoptively transferred to 5-week-old prediabetic NOD mice by i.v. injection in the form of three doses, one week apart, could reduce diabetes incidence from 90% in controls to 20% in the treated cohort at 30 weeks. After i.v. injection, DCs migrated to the spleen, and to a lesser degree to the exocrine tissue of the pancreas, and induced regulatory TH2 cells. This study concluded that DCs prepared in IL-4 may be able to alter the balance between Th1 and Th2 immunity in treated mice. To further examine this hypothesis, a later study (Feili-Hariri et al., Human Gene Therapy (2003) 14:13-23) utilized DCs transduced with adenoviral vectors expressing IL-4 (Ad/IL-4). The DCs migrated to the pancreatic lymph nodes within 24 hours of i.v. administration to NOD mice, but were not detectable after 72 hours. Treatment was most effective when administered to NOD mice at 5 and 8 weeks of age (20-25% of mice treated at 5 weeks with one or two injections of DCs transduced with Ad/IL-4 developed diabetes compared to 70-100% of control mice treated with PBS). However, when treatment was initiated in older pre-diabetic NOD mice (15 weeks of age), a few mice showed delay in onset, but no significant difference in diabetes incidence was observed between treated and PBS control groups.
U.S. Pat. No. 7,378,089 discloses administering dendritic cells and T cells genetically modified to contain an expression cassette encoding a suppressive agent, such as IL-4, for the treatment of autoimmune diseases, including diabetes.
IL-4 has been used to treat animal models of autoimmune diseases other than diabetes. For example, Picarillo and Prud'homme (Hum Gene Ther (1999) 10:1915-1922) disclose that intramuscular injection of a naked plasmid DNA expressing an IL-4/IgG1 chimeric protein protects mice from myelin basic protein-induced experimental allergic encephalomyelitis (EAE), a mouse model for multiple sclerosis. Bessis et al. (J Gene Med (2002) 3:300-307) disclose systemic injection of immortalized fibroblasts transfected with a plasmid encoding IL-4 results in clinical and histological improvement of joint inflammation and destruction in the mouse model of collagen-induced arthritis (CIA). Hogaboam et al. (J Clin Invest (1997) 100:2766-2776) disclose the use of adenoviral-based gene transfer of IL-4 for treatment in an experimental model of inflammatory bowel disease.
Gene therapy approaches using cytokines other than IL-4 have been used in a number of animal models of autoimmune diseases. For example, expression of TGF-β1, IL-10 or galectin-1 is protective in mouse models of diabetes (King et al. (1998) Immunity 8:601-613; Kawamoto et al. Int Immunol (2001) 13:685-695; Perone et al. (2006) J Immunol 117:5278-5289). Costa et al. (J Immunol (2001) 167:2379-2387) disclose the use of myelin basic protein-specific T cells containing a retroviral vector expressing IL-12 p40 for the treatment of EAE, a mouse model for multiple sclerosis.
Guichelaar et al. (J Immunol (2008) 180:1373-1381) disclose cartilage proteoglycan-specific CD4+ T cells transduced with IL-10 for the treatment of proteoglycan-induced arthritis, a mouse model of arthritis. Smith et al. (Gene Ther. (2003) 10:1248-57) disclose the prevention of collagen-induced arthritis in mice by administration of a collagen reactive T-cell hybridoma expressing an anti-TNF single-chain antibody. Transgene expression was detected in the paws but not the spleen of treated animals for up to 55 days postinjection.
U.S. Pat. No. 6,670,186 proposes loading antigen presenting cells with RNA encoding an immunomodulator (i.e., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, or IL-15, or GM-CSF) together with RNA encoding a tumor-derived or pathogen antigen. According to the '186 patent, the RNA-encoded immunomodulator is intended to enhance the immune response.
The immunosuppressive therapies discussed above involve sustained expression or repeated delivery of immunosuppressive cytokines or cytokine expression vectors. However, systemic or prolonged delivery of immunosuppressive cytokines can lead to toxicity, increased risk of infections and malignancies. Accordingly, there is a long-felt need in the art for effective, non-toxic therapies to prevent or treat autoimmune diseases, allergy and transplant rejection.